PROJECT SUMMARY To accommodate the demands of the constantly expanding embryo, vessels of the fetal circulatory system must undergo extensive morphological remodeling while simultaneously supporting pre-existing tissues. Mechanical stimuli, generated by the hemodynamic force, are critical regulators of this process, driving adaptive responses to blood flow such as vessel fusion, widening, or regression. Herein, we propose experiments to determine how early vessels remodel in response to changes in blood flow. Our hypothesis is that differential activation of a Flk1/ERK/Dll4/Notch signaling axis creates a subset of ECs competent to respond to blood flow, resulting in the directed migration and expansion of the vitelline artery. We have assembled an outstanding team of investigators, as well as novel technologies, mouse models and methods to test this hypothesis in three specific aims: 1. Use loss- and gain-of-function models of Flk1/ERK/Dll4/Notch signaling to define differences in endothelial cell (EC) migration during vessel remodeling. 2. Use a real-time ERK biosensor to determine whether ECs that migrate in response to changes in blood flow upregulate ERK activity and if altering ERK signaling, or modulating blood flow, affects cell migration. 3. Employ single cell RNA-sequencing to determine if a specific population of cells exists in the VA with high arterial gene expression and elevated levels of polarization and pro-migratory genes. The transcriptional data will complement the dynamic, 3D imaging data, allowing us to reveal the network of factors required in specialized ECs that would otherwise be lost through studies at a population level (e.g. via bulk RNA-seq) and will no doubt prompt novel future investigation into hemodynamic force signaling and EC migration in the embryo.